• Ape Plasmid Editor Manual Software
• Ape A Plasmid Editor Manual
• Ape Plasmid Editor Manual Download
• Ape Plasmid Software

Based on recent progress in plasmid biology and bioinformatics, we consider three factors that should be taken into account to design a synthetic plasmid : 1) plasmid gene content; 2) interaction with host (host factors and fitness cost imposed by plasmids); and 3) constraints in genome (size, sequence composition e.g., G + C content, oligonucleotide composition, and codon usage, and gene. Most people looking for Ape plasmid editor downloaded: ApE (Universal) Download. 3.6 on 31 votes. ApE is the acronym for A plasmid Editor. Main features: - Highlights restriction sites in the editing window. ApE is an easy to use plasmid editor.

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QIIME (Quantitative Insights Into Microbial Ecology) software
Powerful suite of open source bioinformatics tools for performing microbiome analysis from raw sequences. *RNA

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Information resources

References, texts, and information resources.

Databases

Search engines and data repositories. There are thousands available (see MetaBase). The few items listed here are some of the broadest and most powerful

General Search/Reference

• BioNumbers
  • The database of useful biological numbers. (link to database)

• CDC Disease Conditions
  • Filtered index of diseases from the CDC database

• EMBL-EBI
  • Hugely extensive collection of resources, databases, and tools related to diverse aspects of bioinformatics and molecular biology, often containing everything one might need

• Entrez (NCBI)
  • Search all NCBI databases

• MetaBase
  • The database of biological databases - External link

• Tree of Life
  • Collaborative encyclopedia of biodiversity

• Wolfram Alpha
  • Computational server for finding/calculating summary data about a wide range of topics, plus useful widgets like reagent tables and gene lookup

Genome and Metabolism

• BioCyc
  • Collection of thousands of pathway/genome databases for many organisms, plus software tools for understanding their data

• Colibri by Institut Pasteur
  • E. coli genome browser; get sequences, see the position of your gene in the chromosome, see the function of your gene, and other fun stuff. You can also search for protein sequences/motifs within the E. coli genome.

• EcoGene
  • Database of all E. coli genes and sequences (Entrez data is pulled from here)

• EcoCyc
  • Comprehensive biochemical pathway and gene function site for E. coli

• ENCODE
  • Parts list for functional elements in the human genome

• Ensembl
  • Eukaryotic genome browser

• Ensembl Genome
  • Sister to Ensembl, not limited to Eukaryotes

• Inventory of Composable Elements (ICE) - The public instance of the JBEI Registry
  • A DNA part, plasmid, microbial strain, and Arabidopsis Seed online repository with physical sample tracking capabilities
  • A site where you can explore the various features of the JBEI Registry software, and even get some work done!
  • Try out the integrated online tools, including DNA sequence editing and annotation (Vector Editor) and auto-aligning sequencing trace files against a template.
  • Follow a link to the underlying open-source software source code.

• KEGG
  • Genomic database geared towards high-level functions of the biological system

• Mammalian Gene Collection
  • Access to sequence-validated, full-length, protein-coding, mammalian cDNA clones

• MetaCyc
  • Multi-organismal member of BioCyc collection; catalogs entire universe of metabolism

• SGD
  • Saccharomyces genome database (yeast genome)

• UCSC Genome Bioinformatics
  • Sequence reference for a large number of genomes

Protein

• Antibody Resource
  • Enter a protein to search for antibodies and ELISA kits

• BRENDA
  • Comprehensive enzyme information system

• ConSurf
  • Server for the identification of functional regions in proteins

• Genome3D
  • Consensus structural annotations and 3D models for sequences of model organisms (built upon numerous other useful related resources)

• Technical suppport on protein crystallization by Hampton Research
  • Focused on protein crystallization but contains a lot of generally useful information about various reagents with respect to proteins.

• UniProt
  • Extensive database of protein sequence and functional information

RNA

• Appendix by Ambion, Inc.
  • Website with many useful nucleic acid parameters.

• GreenGenes
  • 16S rRNA database and workbench (most recently updated data can be downloaded here)

• Ribosomal Database Project
  • Quality-controlled, aligned and annotated Bacterial and Archaeal 16S rRNA sequences, and Fungal 28S rRNA sequences, and a suite of analysis tools

• SILVA
  • Massive, searchable rRNA database (especially strong for microbes)

Vectors

• Addgene's Vector DB
  • Addgene is a a non-profit plasmid repository where scientists can archive and share their plasmids. Addgene assists with data submission and all tech transfer issues. Plasmids can be requested from Addgene for a fee to cover expenses.
  • AddGene Vectors can be directly imported into Genome Compiler software platform, in which you can easily edit and visualize it.

Lab Techniques

• Barrick Lab of UT Austin
  • Tons of helpful resources like protocols, guides, links, and tools for lab work

• BenchFly
  • Search, view, upload, create, and host scientific protocol videos

• Current Protocols in Molecular Biology
  • Handbook of protocols. (Links to Wiley Online Library)

• Gene expression in E. coli by Ehrmann lab
  • Tips and information on gene expression in E. coli

• Molecular Cloning by Sambrook and Russell
  • Handbook of protocols. Subscription only. External link

• OWW Materials
  • Useful information for making or obtaining reagents, enzymes, buffers, etc.

• On Being a Scientist by National Academy of Science
  • NAS report on responsible conduct in research.

• OWW Protocols
  • Growing collection of laboratory protocols and techniques

• Polony Protocols by Church and Mitra Lab
  • Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid.

• Protocol-online by Dr. Long-Cheng Li
  • A database of research protocols in a variety of life science fields. It has a popular discussion forum.

• Protocols.io^appweb
  • Huge, open access life science protocol repository for discovery and sharing of scientific methods

• VADLO Search Engine
  • VADLO is a search engine for Life Sciences Protocols, Online Tools, Databases, Software, and Biomedical Powerpoint Lectures. It also has Daily research cartoons, called *'Life in Research' Cartoons.

Microbial physiology

• CyberCell Database (CCDB) by Institute for Biomolecular Design
  • Several tables describing statistical data on E. coli compiled from several sources.

• EcoSal by ASM Press
  • An incomplete web version of the publication Escherichia coli and Salmonella: Cellular and Molecular Biology. (Subscription required)

• Metabolic Pathways Poster PDF by SigmaAldrich
  • Free digital version of the paper Metabolic Pathways map. Because it is a PDF, it is also searchable.

• MicrobeWiki
  • Student edited resource on microbes and microbiology (curated pages reviewed by microbiologists)

Teaching

• Khan Academy (Biology)
  • Selection of science videos and short courses, including the popular Crash Course series

• PhET
  • Interactive simulations for science and math

• ScienceHack
  • Search engine for science videos with a review system

Research Tools

Tools and applications to aid in research tasks.

General

• Benchling^web
  • Online apps for cloning, molecular biology, and analysis

• Bioinformatics Toolbox from DNA2.0 ^web
  • Contains many Javascript tools to do common tasks.

• Cancer Genome Anatomy Project^web
  • Resources and tools related to the characterization of cancer gene expression profiles

• Experiment^web
  • Crowdfunded scientific research

• Genome Compiler^websoftware
  • A powerful, free online & downloadable genetic engineering all-in-one platform for molecular & synthetic biologists

• FastPCR^software
  • An integrated tool for PCR primers or probe design, in silico PCR, oligonucleotide assembly and analyses, alignment and repeat searching

• Galaxy^web
  • Easy-to-use Bioinformatics manipulation tools for UCSC data

• iGEM Parts Registry^web
  • Formerly curated by MIT, this is an open repository of BioBricks; the place for all your standard biological parts.

• Ingeneue by George von Dassow, Eli Meir, Edwin Munro, and Garret Odell at the Center for Cell Dynamics^software
  • An open-source java program for modeling gene regulatory networks. Users can rapidly build networks by specifying their topology, initial conditions, connectivity, and known parameters. Ingeneue can then search/explore paramter space for desired behavior, simulate the effects of noise and mutation, and generate statistics/time graphs of the system.

• MABL^web
  • Reconstruct and analyze phylogenetic relationships between molecular sequences

• OpenCFU^software
  • Local cell colony counting software

• MCDS^software
  • All-in-one molecular cloning and genetic engineering design, simulation & management tool for complex synthetic biology and metabolic engineering project.

• PaR-PaR Laboratory Automation Platform^web
  • PaR-PaR allows researchers to use liquid-handling robots effectively, enabling experiments that would not have been considered previously. After minimal training, a biologist can independently write complicated protocols for a robot within an hour.

• Pinecone by Serotiny ^web
  • High-level design, analysis and transmission of protein constructs. Pinecone matches users' designs with CRO's or DNA synthesizers to produce genetic starting material (dsDNA, plasmid or purified protein). Facilitates mutation and combinatorial protein sets that benefit from manufacturing economies of scale.

Ape Plasmid Editor Manual Software

• PubChase^softwareapp
  • Generates research article recommendations unique to each user. Site also has a special academic community.

• Science Gateway^web
  • Collection of science-related links, including links to journals, catalogs, and tools. See their about page.

Calculators

• [GdmCl] and [Urea] from refractive index by Sosnick lab ^web
  • GdmCl and urea concentration calculator from index of refraction.

• OligoCalc Oligonucleotide Calculator ^web
  • Calculate a number of parameters for your nucleotide polymers

• Statistics to use from Saint John's University. ^web

DNA

• Affymetrix Tools^software
  • Microchip and SNP analysis software. DevNet Tools and other compatible programs also listed for download and use

• Artemis by the Sanger Center ^software
  • A free DNA sequence viewer and annotation tool (Java based). The Sanger Center also develops a number of other miscellaneous tools for download

• BioEdit^software
  • Windows-only sequence alignment editor (no longer maintained, but free to download)

• BLAST^web
  • Finds regions of similarity between biological sequences.

• CLC Sequence Viewer^software
  • Software environment enabling users to make a large number of bioinformatics analyses, combined with smooth data management, graphical viewing, and output options.

• Gene Design by Boeke lab ^web
  • Collection of online tools for codon optimization and shuffling, restriction site editing, and so on.

• GeneDesigner by DNA2.0 ^software
  • Combine genetic building blocks by drag-and-drop, codon optimize, restriction site editing, sequence oligo design etc. See BMC Bioinformatics 2006 Jun 6;7(1):285 for more detail.
  • Free to download and works on Mac or PC. User agreement is somewhat restrictive, i.e. you cannot sell genes designed using the tool without permission.

• GeneWarrior^web
  • Free/simple online toolset for sequence manipulation

• Genome Compiler^websoftware
  • The Complete set of tools for sequence viewing, annotation and alignment. Free online & downloadable and supports all file formats

• IDT SciTools^web
  • A number of web tools and calculators to assist in the design and execution of molecular biology research

• Jalview^software
  • Program for multiple sequence alignment editing, visualization, and analysis

• NEB Cutter by New England Biolabs, Inc. ^web
  • Tool for finding restriction sites, et cetera.

• PerlPrimer^software
  • Open source PCR primer design. Written in Perl/Tk.

• Primer3^web
  • Tool that lets you pick & evaluate primers from a DNA sequence

• QIIME (Quantitative Insights Into Microbial Ecology) ^software
  • Powerful suite of open source bioinformatics tools for performing microbiome analysis from raw sequences.

• Rare Codon Calculator (RaCC) by NIH MBI Laboratory for Structural Genomics and Proteomics ^web
  • Finds rare codons in a coding sequence.

• Search of rare codons in nucleotide sequence by 'Practical Molecular Biology' ^web

• Sequence file format converter from NIH ^web
  • Web tool for converting between sequence file formats.

• Sequence Manipulation Suite^web
  • Quickly access a host of tools for analyzing and manipulating DNA, RNA, and protein sequences

• SIDD^web
  • Stress induced DNA duplex destabilization. Finds destabilized sites in superhelical DNA.

Ape A Plasmid Editor Manual

• UNAFold^web
  • Replacement for mFold for predicting nucleic acid folding. Downloadable and some applications are available online also.

• USEARCH^software
  • High throughput sequence analysis (orders of magnitude faster than BLAST) and other software tools

RNA

• RBS Calculator in Genome Compiler
  • Control translation initiation rate and predict protein production levels.

• Promoter prediction^web
  • both prokaryote and eukaryotic promoter prediction

• UNAfold by Michael Zuker. ^web
  • Apps for predicting RNA and DNA folds, calculating Tm's and free energies. Runs mfold + UNAfold servers

• Sfold^web
  • Statistical Folding and Rational Design of Nucleic Acids. Predicts accessible RNA sites

• Vienna RNA^software
  • RNA secondary structure prediction and design

• XRNA^software
  • Java tools for creating RNA secondary structure diagrams

Protein

• RBS Calculator in Genome Compiler
  • Control translation initiation rate and predict protein production levels

• AGADIR by Serrano lab ^web
  • An algorithm to predict the helical content of peptides.

• AVID by Keating lab ^web
  • An integrative framework for discovering functional relationships among proteins.

• Backbone-dependent rotamer library by Dunbrack lab ^web
  • Libraries of sidechain rotamer from protein structures

• Cn3D by NCBI ^web
  • A helper application for your web browser that allows you to view 3-dimensional structures from NCBI's Entrez retrieval service. It doesn't read PDB files but can be more straightforward to use than DeepView.

• Contact Order Calulator by Baker lab ^web
  • Calculator to determine a protein's contact order

• Dang by Richardsons' lab ^software
  • A command-line tool that generates a table of several useful geometric measurements for each residue or base from a PDB file.

• DeepView by GlaxoSmithKline & Swiss Institute of Bioinformatics ^software
  • Awesome program for viewing and studying protein structure.

• ExPASy Proteomics server by the Swiss Institute of Bioinformatics ^web
  • Collection of links to many pages to calculate parameters of your favorite proteins
  1. Compute pI/Mw
     • A tool which allows the computation of the theoretical pI (isoelectric point) and Mw (molecular weight) for a list of Swiss-Prot and/or TrEMBL entries or for user entered sequences.
  2. ProtParam
     • A tool which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence.
  3. Translate
     • Translate is a tool which allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence.

• GETAREA by Sealy Center for Structural Biology ^web
  • Solvent accessible surface areas, atomic solvation energies, and their gradients for macromolecules

• Metazome^web
  • Proteome-level phylogeny and genomics

• Modeller by Sali lab ^software
  • Program for homology or comparative modeling of protein three-dimensional structures by satisfaction of spatial restraints.

• PAIRCOIL2 by Keating and Berger labs ^web
  • Tool to predict the parallel coiled coil fold from sequence using pairwise residue probabilities.

• ProteinProspector by UCSF Mass Spectrometry Facility ^web
  • Proteomics tools for mining sequence databases in conjunction with Mass Spectrometry experiments.

• PyMOL^software
  • Molecular graphics system with an embedded Python interpreter designed for real-time visualization and rapid generation of high-quality molecular graphics images and animations. The latest version does not run on OSX 10.3. (from Kathleen).

• VMD by Theoretical and Computational Biophysics Group at UIUC ^software
  • Molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting. Generates pretty high resolution pictures of protein structures.

• Zinc Finger Tools by Barbas lab ^web
  • Design Zinc Finger DNA binding proteins

Vectors

• ApE A plasmid Editor ^software
  • Simple tools for plasmid design and manipulation

• GeneDesigner by DNA 2.0 ^software
  • Design genes de novo with a powerful and intuitive user interface

• Genome Compiler^websoftware
  • A free all-in-one platform for intuitive vector design and automatic assembly. Supports main construction techniques, manual and auto sequence annotation, alignment, primer design and compatible to all file formats.

• j5, DeviceEditor, and VectorEditor^web
  • j5: DNA assembly design automation for (combinatorial) flanking homology (e.g., SLIC/Gibson/CPEC/SLiCE/yeast) and type IIs-mediated (e.g., Golden Gate/FX cloning) assembly methods
  • DeviceEditor: a visual DNA design canvas that serves as front-end for j5
  • VectorEditor: a visual DNA editing and annotation tool

• PlasMapper^web
  • Generates and annotates a plasmid map based on sequence data

• Serial Cloner^software
  • Light and intuitive tools for diverse molecular biology manipulations

• SnapGene Viewer^software
  • Free version of SnapGene. Allows mapping of DNA up to 1 Gb in length. Convenient viewing and annotation tools for genetic constructs.

• Vector NTI^software
  • Powerful but expensive vector editor. Supports most sequence types and easily manipulates genetic constructs.

Writing/Composition/Organization

• CambridgeSoft^software
  • Numerous proprietary applications for research and composition (ChemBioDraw is usually accessible via academic institution email)

• Coredemia^web
  • Community for sharing and discussion of research papers

• Google Documents by Google^web
  • Sharable, web-based word processing, spreadsheets, and presentations

• Evernote^softwareapp
  • Powerful cross-platform note-taking, composition, and organization with cloud syncing.
• InstaGrok^software
  • Tool for seeking, collecting, and mapping information

• Inkscape^web
  • Free, feature-rich drawing program with support for SVG and PDF images

• Mendeley^softwareapp
  • Powerful and straightforward local and cloud-based reference manager

• Origin^software
  • Scientific graphing and data analysis software

• Qiqqa^software
  • Organize, annotate, search, and cite references from a PDF library (loads of extra features)

• ReadCube^software
  • Reference manager, similar to Mendeley or Evernote. Can enhance PDFs and search PubMed from within the app.

• ThinkFree Office Online by ThinkFree ^web
  • Beta suite of office applicatins offered over the web

• Vessel^webapp
  • Digital lab notebook for organizing and sharing experiments, findings, protocols, and more.

• WriteBoard by 37signals ^web
  • Permits writing of shareable, web-based text documents

• Writely by Upstartle, LLC. ^web
  • A beta word processor run over the web

• Zoho^web
  • Sharable, web-based word processing, spreadsheets, and presentations (alternative to Google Docs)

See also:

Vectors - info/links related to cloning vectors.

Searching the literature - info/links on searching the scientific literature.

Synthetic Biology - info/links on Synthetic Biology.

Here are optimized methods and tips from the Chien lab, adopted after trial and error, which work consistently in our hands. See the Invitrogen multisite Gateway manual for all of the basic information necessary to understand and perform Gateway recombination reactions. See also the Gateway tips on the Lawson lab website.

Please report problems or questions on the Tol2kit blog.

As a first test, we suggest that you grow up entry clones and perform a test LR reaction with pDestTol2pA2 or pDestTol2CG2 to make an expression clone such as bactin2:EGFP-pA.

• 3BP Reactions
• 4LR reactions
• 5Assembling sequences for expression clones
• 6Injections for transgenesis

Growing up clones

Entry clones are kanamycin-resistant and can be transformed and grown in any standard E. coli strain. For a single lab's use, minipreps (e.g. using Qiagen miniprep columns) are sufficient for the entry vectors; you will use very little DNA for each LR reaction. If you run low, you can always just miniprep again.

However, note that destination vectors have an ampicillin resistance gene in the backbone, donor vectors have a kanamycin resistance gene in the backbone, and both have a ccdB suicide gene and a chloramphenicol resistance gene in the 'gate' (the ccdB provides negative selection during the BP or LR reaction). Therefore these clones must be grown in ampicillin/chloramphenicol or kanamycin/chloramphenicol, in ccdB-tolerant cells (available from Invitrogen). In addition, we have found that certain destination vectors and donor vectors are prone to recombination or mutation, so at a minimum you should test each DNA prep by careful restriction analysis. We have also found that while propagating these vectors, it is crucial to pour plates of the desired antibiotic resistance; it is not sufficient to pipet chloramphenicol solution onto a pre-poured ampicillin or kanamycin plate.

For donor vectors and destination vectors, which you will use repeatedly and which are tricky to grow up correctly, we recommend that you grow at least a midiprep or maxiprep scale.

We use the following antibiotic concentrations: ampicillin (100 ug/ml), kanamycin (50 ug/ml), and chloramphenicol (30 ug/ml), both for selection on plates as well as in liquid culture.

What you will need

Here are the specific reagents required for working with the Tol2Kit, including Invitrogen catalog numbers. Where more than one catalog number is listed, this reflects the different sizes available.

BP Clonase II Enzyme Mix (11789-020, 11789-100): for generating entry clones. Note: unlike BP Clonase, which had a separate buffer, BP Clonase II includes the buffer in the enzyme mix.

Ape Plasmid Editor Manual Download

LR Clonase II Plus Enzyme Mix (12538-120, 12538-200): for generating expression constructs via the multi-site reaction. Note: LR Clonase II (no Plus) is a different enzyme, to be used for 'classic' (non-multisite) Gateway reactions. Note: make sure to store LR Clonase II Plus at -80 degrees, as it seems to be especially labile. (We usually also store the BP Clonase II and of course the One Shot cells at -80 degrees.)

One Shot ccdB Survival Competent Cells (C7510-03): for propagation of empty donor and destination vectors.

One Shot TOP10 Chemically Competent E. coli (C4040-10, C4040-03, C4040-06): for transformation of LR reactions. Note: do NOT use One Shot TOP10F' cells; these will not show a difference in clear and opaque colonies (see below).

pDONR221 (12536-017): empty middle entry vector for generation of new middle clones.

pDONR P4-P1R and pDONR P2R-P3 (12537-023): empty 5' and 3' donor vectors for generation of new 5' and 3' entry clones. Note: the catalog number provided here is for the MultiSite Gateway Three-Fragment Vector Construction Kit; it includes pDONR221 as well as a destination vector (pDest R4-R3). This appears to be the only way to purchase the empty donor vectors at this point.

Note that the Tol2kit is based on the original three-part multisite Gateway system (as described in the version D manual), in which destination vectors use attR4-R3 sites, not the new Multisite Gateway Pro system, in which all the destination vectors use attR1-R2 sites. While the donor, entry, and destination vectors are incompatible with the Pro system (different sets of att sites are used), the BP and LR enzyme mixes are still the same.

BP Reactions

Donor Vectors

The Invitrogen multisite Gateway manual describes and explains the donor vectors in detail. The one extra technical note is that the 5' donor vector (pDONR P4-P1R) can be tricky to propagate due to self-recombination. Based on advice from the Lawson lab, we now use their 5' donor vector propagation protocol.

PCR Amplification of DNA

Primers for PCR are designed as described in the multisite Gateway Manual. This results in primers that are quite long (regularly >50 bases), but we have not had difficulty performing PCR with these primers. This list of att site sequences may be useful.

We have used two different polymerases for PCR: Tth (GeneAmp XL PCR Kit; Applied Biosystems) and Phusion (NEB). Both are proofreading polymerases that can amplify long pieces of DNA, although for particularly difficult and/or long promoters, Phusion has worked better. For each, PCR was performed in a 50 ul reaction.

Purification of PCR products

The entire PCR reaction (50 ul) is loaded onto an agarose gel. The appropriate band is excised and DNA purification performed using the Qiagen Qiaquick Gel Extraction Kit (for DNA fragments <10 kb; for larger fragments, use the QIAEX Gel Extraction Kit). Elute the DNA in 30 ul (the smallest recommended volume). The concentration of DNA is calculated using a spectrophotometer; the DNA will be quite dilute and not terribly clean (usually between 10-80 ng/ul, and OD 260/280 ~1.4-1.6).

Do not let the gel-purified DNA sit in the freezer for too long before using in the recombination reaction. In practice, we go straight into the recombination reaction; a better stopping point is to freeze either the entire PCR reaction or the gel slice before purification. We have found that storing the DNA in the freezer for even a couple of days decreases the efficiency of recombination.

BP Recombination Reactions

The recombination reaction is performed as described in the Invitrogen Multi-Site Gateway Manual. An equimolar amount of the appropriate donor vector and purified PCR product (commonly 50-100 femtomoles) are combined with TE and BP Clonase II enzyme mix in a final volume of 10 ul. This reaction is usually allowed to incubate overnight at room temperation, however, we have found (as the manual also suggests) that as little as 2 hours can be enough. This reaction almost always works well. Note that the suggested reaction volume of 10 ul is the 'full' reaction suggested by the Invitrogen manual. Other labs have moved to setting up half-reactions (5 ul final volume); this works as well.

Transformation, Plasmid Prep, and Diagnostic Digests

The BP reaction is treated with Proteinase K and transformed. Typically, 2 ul of the 10 ul reaction is sufficient. The Invitrogen Manual recommends OneShot TOP10 cells, but cells of this high competence are not necessary. Subcloning efficiency cells and also homemade competent cells have worked well, yielding hundreds to thousands of colonies per plate. We typically pick 4 colonies for minipreps, and these are almost always the correct clone. Check by restriction, and then by sequencing for PCR errors. For restriction tests, we try to pick enzymes that do not cut in the att sites. PvuII is often useful, as are EcoRV and HindIII.

Note: the clear/opaque difference in colonies applies only to transformants from the LR reaction, not this (the BP) reaction.

LR reactions

Recombination Reactions

The recombination reaction is performed with slight modifications from the protocol in the Invitrogen Multi-Site Gateway manual. Equimolar amounts of entry vectors (5', middle, and 3') and destination vector are combined with LR Clonase II Plus enzyme mix. Note that unlike the earlier version (LR Clonase Plus), there is no separate buffer (it comes premixed with the enzyme). We standardly set up reactions with 20 femtomoles of each vector in a 10 ul reaction. The original manual has a protocol for a 20 ul reaction, however, this enzyme mix (LR Clonase II Plus) comes with a protocol for a 10 ul reaction. Other labs have found that half reactions (5 ul) work as well.

We always allow this reaction to go overnight at room temperature. The reaction tends to be less efficient than the BP reaction, likely because of the number of components involved.

Transformation, Plasmid Prep, and Diagnostic Digests

As with the BP reaction, the LR reaction is treated with Proteinase K and then transformed. We typically transform 3 ul of the 10 ul reaction, using Invitrogen OneShot TOP10 cells. Because this reaction is less efficient than the BP reaction, cells of this high competence are necessary. The protocol for these cells recommends shaking at 37 degrees for one hour after heat shock. Instead, we generally shake for 1.5 hours, just to give the cells more time to grow before selection. We then plate all 300 ul onto the LB/amp plate. Particular LR recombination reactions can be less efficient than others (see below), and we believe that giving the culture one more doubling time will increase the chance that the correct clone can be isolated.

This reaction is plated onto ampicillin plates; carbenicillin works as well. We typically find hundreds of colonies per plate. We do not plate the reaction before 3 pm, as satellite colonies can be a significant problem, obscuring the results of the reaction. Plates are removed from the 37 degree incubator first thing the next morning; this provides the best chance to distinguish clear from opaque colonies. If it is difficult to tell clear from opaque, looking at the plate in front of a dark background (we use a black refrigerator) will help. The image below shows examples of clear and opaque colonies on the same plate.

Plates can be left at room temperature until clear colonies are picked in the afternoon. We have found that clear colonies contain the correct clone >99% of the time, while opaque colonies never contain the correct clone. A reaction that has worked well will have a clear to opaque colony ratio of at least 3:1. However, as long as clear colonies can be identified, the correct clone will be isolated. As with the BP reaction, clones are tested via restriction digest; again, we generally avoid enzymes that cut within the att sites. PvuII has been very useful for this.

Factors Affecting Reaction Efficiency

Certain entry vectors seem to be less efficiently recombined in the LR recombination reaction. The lower efficiency of the reaction will be conveyed by a lower number of transformants, as well as a lower ratio of clear to opaque colonies. The major factor leading to lower recombination efficiency appears to be size of the insert. Both particularly short and extremely long DNA fragments can be tricky. Short is defined as less than 200 bases (e.g. p3E-polyA in the Tol2Kit), and long is defined as greater than 10 kb. Although these reactions work less efficiently than others, we have not defined a lower limit for fragment length. For example, p5E-Fse-Asc has an insert of 59 bases; this will still work in recombination reactions. On the other hand, entry vector fragments of greater than 10 kb have been difficult to work with; it is not clear if this reflects something about the recombination reaction or the specific DNA insert.

Assembling sequences for expression clones

Each of the 4 sets of att sites has a core sequence, e.g. 'attB4/L4/R4_shared', which is shared between the attL, R, B, and P sites (see sequences here). This means that adjacent clones used to build an expression clone will have at least a 15 bp overlap at the ends. This overlap can be used to predict the sequence of the expression clone, for instance to pick diagnostic restriction digests.

by hand or with Sequencher

Here are relevant sequences (entry clone inserts, destination clone ends) and a detailed description of how to build expression clone sequences using a simple sequence assembly program like Sequencher.

using ApE

12/18/07: We have now started using Wayne Davis' really lovely shareware program [ApE] ('A plasmid Editor'), which can calculate Gateway recombinations for you:

• Open sequences for the three entry clones and destination clone (e.g. using the Genbank-format sequences provided on the wiki). (Make sure that all sequences are marked as circular, not linear.)
• Select Tool>Recombination Tool..., and select the multisite Gateway prototype.
• Hey presto, you have your expression clone sequence.

ApE does lots of other things too--think DNA Strider on steroids. Thanks Wayne!

sequence deviations

When sequencing entry clones, you may occasionally notice (as we have) a single base in an att site differing from that given by Invitrogen. Apparently, their documented sequence is not base-perfect. We will list these differences here as we notice them.

C>A change (shown in lowercase here) in the attL1 and attP1 sequences:

Injections for transgenesis

Preparation of capped transposase RNA

pCS2FA-transposase is linearized using NotI and purified using the Qiagen PCR Purification Kit. In vitro transcription is carried out using the Ambion mMessage mMachine SP6 Kit (catalog #1340); 2 ug linearized DNA is used in each transcription reaction. In vitro transcribed RNA is purified using the Qiagen RNeasy Mini Kit, and subsequently ethanol precipitated and resuspended in a final volume of 20 ul. The RNA concentration is then quantified using a spectrophotometer, and run on a gel to confirm its integrity. We generally get 20 ul of approximately 1 ug/ul capped RNA from each reaction.

Ape Plasmid Software

Injections

Injections are performed at the 1-cell stage. It is crucial to inject the nucleic acids into the cell (not the yolk); this most increases the chance of early integration.

Spotting DNA for distribution

For distribution, we usually dilute maxiprep DNA to 250 ng/ul in 0.025% bromphenol blue (using a 10x stock of 0.25% BPB in TE), then spot 2 ul per construct onto Whatman paper (final amount 500 ng). In cases where the maxiprep concentration was low, we have spotted as little as 125 ng.